Our major goal is to identify biologically relevant pathways that can explain the high frequency of mutations at methylated CpG (mCpG) sequences in the p53 gene and other genes. It is an almost universally accepted dogma that these mutations are caused by spontaneous deamination of 5-methylcytosine. However, it can be calculated that only a few 5-methylcytosines deaminate spontaneously in double-stranded DNA per day in each cell. Our hypothesis is that mCpG transitions are not caused simply by deamination of 5-methylcytosine in double-stranded DNA but by other processes including, for example, mCpG-specific base modification by endogenous or exogenous mutagens, or, alternatively, by secondary factors operating at mCpG sequences and promoting deamination. We will test if oxidative and/or nitrosative DNA damage can cause C to T transition mutations at mCpG sequences preferentially. We will test if deamination of 5-methylcytosine glycol in oxidized DNA plays a role in this process. Similarly, we will test if agents that produce exocyclic (etheno) DNA adducts can preferentially target 5-methylcytosine. The effects of nitric oxide on mCpG mutagenesis will be tested using in vitro and in vivo systems. To support the in vitro mutagenesis studies, we will use bacteriophage lambda transgenic mice on a genetic background with manipulated levels of reactive oxygen species in order to elucidate the mutational consequences of in vivo oxidative stress in chromosomal reporter genes with high levels of CpG methylation. We will also test if deamination of 5-methylcytosine is enhanced by secondary factors that operate at mCpG sequences. Mammalian cells contain at least five proteins that can bind specifically to methylated CpGs. We will test if binding of these proteins to CpG-methylated DNA enhances deamination of 5-methylcytosine, alters the chemical reactivity of mCpG sequences towards endogenous or exogenous mutagens and/or affects repair and mutagenesis at these sites. In addition, we will investigate the role of AID and APOBEC homologues in mCpG mutagenesis. These approaches should increase our understanding of the basic mechanisms leading to the most common type of single base substitution in mammalian cells.